PROJECT SUMMARY Infective endocarditis (IE) is a serious and often deadly disease in which bacteria colonize the heart valves and cause severe damage. The exact prevalence of the illness has been difficult to define due to diagnostic challenges; however, of the thousands of cases diagnosed annually around the world, as many as 40% of them are fatal. The high mortality rate of IE is due mostly to ineffective treatment options, which are currently limited to aggressive antibiotic treatments and cardiac surgeries. IE caused by the Gram-positive bacteria Staphylococcus aureus has been the deadliest and most common form of IE for over a decade. Recent clinical data demonstrate a correlation between platelet levels in circulation and severity of IE, as patients who develop thrombocytopenia, or low platelet levels, have lower chances of survival compared to patients with normal platelet levels. Additionally, S. aureus expresses two proteins, von Willebrand factor binding protein (VWbp) and staphylococcal protein A (SPA), that bind von Willebrand factor (VWF), a large plasma protein that mediates platelet adherence to the endothelium and facilitates platelet clearance from the bloodstream. Animal models in which VWF is absent exhibit reduced bacterial colonization in both the heart and endothelial tissue. Clinical data and basic research have therefore implicated VWF as a critical mediator in the early development and later progression of IE, yet the molecular mechanism underlying this role of VWF is unknown. Recently, our lab identified the autoinhibitory module (AIM) in VWF that limits its association to platelets. Our lab has also elucidated the molecular mechanisms of platelet activation and clearance when VWF binds and stimulates platelet receptor GPIba under shear flow. Aberrant binding of VWF to GPIba can lead to enhanced platelet activation and cause thrombosis or increased platelet clearance which leads to thrombocytopenia. We hypothesize that S. aureus proteins SPA and VWbp facilitate early bacterial colonization and later thrombocytopenic conditions by activating VWF to enhance platelet binding and yield platelet activation and clearance. In Specific Aim 1, we will determine how SPA and VWbp affect VWF autoinhibition by assessing changes in binding and AIM dynamics. In Specific Aim 2, we will investigate how these S. aureus proteins modulate VWF-mediated platelet activation by assessing binding of VWF and GPIba in the presence of these proteins, measuring hallmarks of platelet clearance signals via flow cytometry, and determining the ability of these proteins to facilitate platelet clearance in mice. My proposed studies will elucidate a molecular mechanism underlying S. aureus pathogenesis and IE development to facilitate potential therapeutic strategies as well as contribute to our fundamental understanding of hemostatic and thrombotic regulation during bacterial infection.